Reconstituted HDL Formulation

ABSTRACT

The present invention relates to reconstituted high density lipoprotein (rHDL) formulations comprising an apolipoprotein, a lipid and a lyophilization stabilizer. Said formulations have reduced renal toxicity and good long-term stability, especially in lyophilized form.

TECHNICAL FIELD

The present invention relates to reconstituted high density lipoproteinformulations, and in particular to formulations with suitable stabilityand biological properties for pharmaceutical use.

BACKGROUND ART

High-density lipoproteins (HDLs) form a range of lipoprotein particlesfound in normal serum. Mature HDL particles are present in the form of aglobular structure containing proteins and lipids. Within the outerlayer of these particles are the more polar lipids, phospholipids andfree cholesterol, all having charged groups orientated outwards, towardsthe aqueous environment. The more hydrophobic lipids, such as esterifiedcholesterol and triglycerides, reside in the core of the particle. Newlyformed or nascent HDL particles lack the lipid and are discoidal inshape. Protein components are embedded in the outer layer. The mainprotein component is apolipoprotein A-I (Apo A-I) with smaller amountsof Apo A-II, Apo A-IV, Apo CIII, Apo D, Apo E and Apo J. Various otherproteins reside on the HDL particle, such as lecithin-cholesterol acetyltransferase, PAF acetylhydrolase and paraoxonase. HDLs are characterizedby high density (>1.063 g/ml) and small size (Stoke's diameter=5 to 17nm).

Efforts have been made to develop artificial HDLs that can be infusedinto the bloodstream of patients to mimic the biological effects ofnaturally-occurring HDLs. These artificial particles are generallyreferred to as “reconstituted HDL” (rHDL), or sometimes as HDL mimeticsor synthetic HDL particles. The artificial particles contain componentsof the natural particles, in particular Apo A-I and lipids. For example,WO 2012/000048 describes rHDL comprising Apo A-I, phosphatidylcholine(PC) and a small amount of sodium cholate. WO 2012/109162 describes rHDLcomprising Apo A-I, sphingomyelin (SM) and phosphatidylglycerol (e.g.1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG)).

It is convenient for rHDL formulations to be lyophilized (freeze-dried)before use. Lyophilization is a commonly used method for preparing solidprotein pharmaceuticals. However, this process generates a variety offreezing and drying stresses, such as concentration of the solubilizedprotein, formation of ice crystals, pH changes, etc. All of thesestresses can denature proteins to various degrees. Thus, stabilizers areoften required in a protein formulation to protect protein stabilityboth during freezing and drying processes. In order to maintain thestability of rHDL formulations during lyophilization, stabilizers likesugars and sugar alcohols have been used. For example, U.S. Pat. No.5,089,602 discloses plasma-derived lipoproteins that are stabilized with10% sucrose or a mixture of 10% sucrose and 5% mannitol. WO 2012/000048discloses sugar and sugar alcohol stabilizers used at a concentrationfrom about 65 to 85 g/L of rHDL formulation (equivalent to about 6.5 to8.5% w/w). WO 2012/109162 discloses sucrose and mannitol as stabilizers,used in a mixture at 4% w/w and 2% w/w respectively. An investigationinto the manufacturing and shelf stability of rHDL was carried out inKim et al, Biotechnology and Bioprocess Engineering 16, 785-792 (2011).Here, rHDL with an Apo A-I:soybean PC ratio of 1:150 could not besufficiently stabilized with 1 or 5% sucrose, whereas 10% sucrose wasdescribed as optimal.

The rHDL formulations of these documents are intended for infusiontherapy, but high sugar concentrations in infusion products may cause orexacerbate renal problems. This is a particular problem in the targetpatient population for rHDL, because these patients are often renallyimpaired.

Therefore, an object of the present invention was to provide alternativeor improved rHDL formulations compared to these previous formulations.In particular, the inventors sought stable rHDL formulations withreduced renal toxicity.

This problem is solved by the formulation according to claim 1. Furtherpreferred embodiments are defined in the dependent claims.

Surprisingly, it has been found that the rHDL formulation of claim 1shows good long-term stability. By containing less lyophilizationstabilizer than previous formulations, the formulation also presentsless risk of renal toxicity. The low lyophilization stabilizerconcentration may also allow the rHDL to perform better in functionalassays of rHDL function. The inventors have also found that amino acids,particularly proline, are useful lyophilization stabilizers for rHDLformulations.

SUMMARY OF THE INVENTION

The invention provides an rHDL formulation comprising an apolipoprotein,a lipid and a lyophilization stabilizer, wherein the ratio between theapolipoprotein and the lipid is from about 1:20 to about 1:120(mol:mol).

Preferably, the lyophilization stabilizer is present in a concentrationfrom about 1.0% to about 6.0% (w/w of rHDL, formulation), e.g. from 1.0,1.1, 1.2 or 1.3 to 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. This low amount oflyophilization stabilizer may reduce the risk of renal toxicity. It isalso particularly suitable for patients receiving contrast agents duringacute coronary syndrome therapy (ACS), since these agents may competewith lyophilization stabilizer for clearance in the kidneys. In apreferred embodiment, the lyophilization stabilizer is present in aconcentration from about 1.0% to less than 6.0% e.g. from about 1.0% to5.9%. Preferably the lyophilization stabilizer is present in aconcentration from about 3.0 to less than 6.0%, e.g. from about 3.0 to5.9%. More preferably, the lyophilization stabilizer is present in aconcentration from about 4.0 to 5.5%, particularly 4.3 to 5.3%, moreparticularly 4.3 to 5.0%, and most preferably 4.6 to 4.8% (w/w). Suchformulations show good stability and low renal toxicity.

Alternatively, or in addition, it is preferred for the ratio between theapolipoprotein and the lyophilization stabilizer to be from about 1:1 toabout 1:3 (w:w). In particular, the ratio between the apolipoprotein andthe lyophilization stabilizer is from about 1:1 to about 1:2.4 (w:w),e.g. less than 1:2 (w:w). The inventors have found that theseformulations remain stable showing few or no changes in the sizedistribution of lyophilized samples, even after storage for severalmonths. However, in some embodiments, the ratio between theapolipoprotein and the lyophilization stabilizer may be less than this,e.g. from about 1:1 to about 1:7, and in particular from about 1:1 toabout 1:5 (w:w).

The invention also provides an rHDL formulation comprising anapolipoprotein, a lipid and a lyophilization stabilizer, wherein thelyophilization stabilizer comprises an amino acid. Preferably the aminoacid is proline. The inventors have found that amino acids are goodlyophilization stabilizers for rHDL formulations, particularly when in amixture with low amounts of other stabilizers.

The invention also provides the aforementioned rHDL formulation forpreventing or treating a disease, disorder or condition in a human.Suitably, the disease, disorder or condition is responsive toprophylactic or therapeutic administration of the rHDL formulation.

DETAILED DESCRIPTION OF THE INVENTION

Within the context of the present invention, the term “reconstituted HDL(rHDL) formulation” means any artificially-produced lipoproteinformulation or composition that is functionally similar to, analogousto, corresponds to, or mimics, high density lipoprotein (HDL), typicallypresent in blood plasma. rHDL formulations include within their scope“HDL mimetics” and “synthetic HDL particles”.

Within the context of the present invention, the term “lyophilizationstabilizer” means a substance that stabilizes protein duringlyophilization. Such lyophilization stabilizers are well known in theart and are reviewed in, for example, Wang (2000) International Journalof Pharmaceuticals 203:1-60. A preferred lyophilization stabilizer foruse in the invention comprises a sugar, a sugar alcohol, an amino acid,or a mixture thereof. For example, the inventors have found thatdisaccharides such as sucrose are particularly suitable sugars for useas the lyophilization stabilizer. Other disaccharides that may be usedinclude fructose, trehalose, maltose and lactose. In addition todisaccharides, trisaccharides like raffinose and maltotriose may beused. Larger oligosaccharides may also be suitable, e.g. maltopentaose,maltohexaose and maltoheptaose. Alternatively, monosaccharides likeglucose, mannose and galactose may be used. These mono-, di-, tri- andlarger oligo-saccharides may be used either alone or in combination witheach other. As noted above, lyophilization stabilizers that are sugaralcohols may also be used. These sugar alcohols may also be used eitheralone or in combination. A particular sugar alcohol for use in theinvention is mannitol. Other sugar alcohols that may be used includeinositol, xylitol, galactitol, and sorbitol. Other polyols like glycerolmay also be suitable. Amino acids that may be used as lyophilizationstabilizers include proline, glycine, serine, alanine, and lysine.Modified amino acids may also be used, for example 4-hydroxyproline,L-serine, sodium glutamate, sarcosine, and y-aminobutyric acid. Theinventors have found that proline is a particularly suitable amino acidfor use as a lyophilization stabilizer.

In particular embodiments, the lyophilization stabilizer comprises amixture of a sugar and a sugar alcohol. For example, a mixture ofsucrose and mannitol may be used. The sugar and the sugar alcohol may bemixed in any suitable ratio, e.g. from about 1:1 (w:w) to about 3:1(w:w), and in particular about 2:1 (w:w). Ratios less than 2:1 areparticularly envisaged, e.g. less than 3:2. Typically, the ratio isgreater than 1:5, e.g. greater than 1:2 (w:w). In some embodiments theformulation comprises less than 4% sucrose and 2% mannitol (w/w of rHDLformulation), for example 3% sucrose and 2% mannitol. In someembodiments the formulation comprises 4% sucrose and less than 2%mannitol. In some embodiments the formulation comprises less than 4%sucrose and less than 2% mannitol e.g. about 1.0% to 3.9% sucrose andabout 1.0% to 1.9% (w/w) mannitol.

In particular embodiments, the lyophilization stabilizer comprises amixture of a sugar and an amino acid. For example, a mixture of sucroseand proline may be used. The sugar and the amino acid may be mixed inany suitable ratio, e.g. from about 1:1 to about 3:1 (w:w), and inparticular about 2:1 (w:w). Ratios less than 2:1 are particularlyenvisaged, e.g. less than 3:2 (w:w). Typically, the ratio is greaterthan 1:5, e.g. greater than 1:2 (w:w). Preferably the amino acid ispresent in a concentration of from about 1.0 to about 2.5% e.g. from1.0, 1.2, or 1.3 to 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5% (w/w of rHDLformulation). In some embodiments the formulation comprises 1.0% sucroseand 2.2% proline, or 3.0% sucrose and 1.5% proline, or 4% sucrose and1.2% proline. The amino acid may be added to the sugar to maintain anisotonic solution. Solutions with an osmolality of greater than 350mosmol/kg are typically hypertonic, while those of less than 250mosmol/kg are typically hypotonic. Solutions with an osmolality of from250 mosmol/kg to 350 mosmol/kg are typically isotonic.

In particular embodiments, the lyophilization stabilizer comprises amixture of a sugar alcohol and an amino acid. The lyophilizationstabilizer may comprise a mixture of a sugar, a sugar alcohol, and anamino acid.

The apolipoprotein may be any apolipoprotein which is a functional,biologically active component of naturally-occurring HDL or of areconstituted high density lipoprotein/rHDL. Typically, theapolipoprotein is either a plasma-derived or recombinant apolipoproteinsuch as Apo A-I, Apo A-II, Apo A-V, pro-Apo A-I or a variant such as ApoA-I Milano. Preferably, the apolipoprotein is Apo A-I. More preferablythe Apo A-I is either recombinantly derived comprising a wild typesequence or the Milano sequence or alternatively it is purified fromhuman plasma. The apolipoprotein may be in the form of abiologically-active fragment of apolipoprotein. Such fragments may benaturally-occurring, chemically synthetized or recombinant. By way ofexample only, a biologically-active fragment of Apo A-I preferably hasat least 50%, 60%, 70%, 80%, 90% or 95% to 100% or even greater than100% of the lecithin-cholesterol acyltransferase (LCAT) stimulatoryactivity of Apo A-I.

In the present invention the molar ratio of apolipoprotein:lipid istypically from about 1:20 to about 1:120, and preferably from about 1:20to about 1:100, more preferably from about 1:20 to about 1:75 (mol:mol),and in particular from 1:45 to 1:65. This range includes molar ratiossuch as about 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65,1:70, 1:75, 1:80, 1:85, 1:90, 1:95 and 1:100. A particularlyadvantageous ratio of apolipoprotein:lipid is from 1:40 to 1:65(mol:mol). This ensures that the rHDL formulation according to thepresent invention comprises a lipid at a level which does not causeliver toxicity.

In other embodiments, the molar ratio of apolipoprotein:lipid may be ina range from about 1:80 to about 1:120. For example, the ratio may befrom 1:100 to 1:115, or from 1:105 to 1:110. In these embodiments, themolar ratio may be for example from 1:80 to 1:90, from 1:90 to 1:100, orfrom 1:100 to 1:110. In a preferred embodiment the rHDL formulationaccording to the present invention comprises additionally a detergent inorder to further stabilize the rHDL particles. The detergent may be anyionic (e.g. cationic, anionic, zwitterionic) detergent or non-ionicdetergent, inclusive of bile acids and salts thereof, suitable for usein rHDL formulations. Ionic detergents may include bile acids and saltsthereof, polysorbates (e.g. PS80),3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate-(CHAPS),3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), cetyl trimethyl-ammonium bromide, lauroylsarcosine, tert-octylphenyl propanesulfonic acid and 4′-amino-7-benzamido-taurocholic acid.

Bile acids are typically dihydroxylated or trihydroxylated steroids with24 carbons, including cholic acid, deoxycholic acid, chenodeoxycholicacid or ursodeoxycholic acid. Preferably, the detergent is a bile saltsuch as a cholate, deoxycholate, chenodeoxycholate or ursodeoxycholatesalt. A particularly preferred detergent is sodium cholate. Theconcentration of the detergent, in particular of sodium cholate, ispreferably 0.3 to 1.5 mg/mL. The bile acid concentration can bedetermined using various methods including colorimetric assay (forexample, see Lerch et. al., 1996, Vox Sang. 71:155-164; Sharma, 2012,Int. J. Pharm Biomed. 3(2), 28-34; & Gallsäuren test kit andGallsauren-Stoppreagens (Trinity Biotech)). In some embodiments of theinvention the rHDL formulation comprises cholate levels of 0.5 to 1.5mg/ml, as determined by colorimetric assay and a lyophilizationstabilizer in a concentration from about 4.0 to 5.5%, particularly 4.3to 5.3%, more particularly 4.3 to 5.0%, and most preferably 4.6 to 4.8%(w/w). In particular embodiments the lyophilization stabilizer issucrose. Such formulations show good stability and low renal and livertoxicity.

The ratio between the apolipoprotein and the lyophilization stabilizeris usually adjusted so this ratio is from about 1:1 to about 1:7 (w:w).More preferably, the ratio is from about 1:1 to about 1:3, in particularabout 1:1.1 to about 1:2. In specific embodiments the rHDL formulationsthus have ratios of 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7,1:1.8, 1:1.9 or 1:2 (w:w). It is however contemplated that forparticular embodiments where there are low amounts of protein (e.g.<20mg/mL) that the ratio between the apolipoprotein and thelyophilization stabilizer can be extended to as much as about 1:7 (w:w),e.g. about 1:4.5 (w:w).

Suitably, the apolipoprotein is at a concentration from about 5 to about50 mg/ml. This includes 5, 8, 10, 15, 20, 25, 30, 35, 40, 45 and 50mg/ml and any ranges between these amounts. The apolipoprotein is,preferably, at a concentration from about 25 to 45 mg/ml. In otherembodiments, the apolipoprotein may be at a concentration of from about5 to 20 mg/ml, e.g. about 8 to 12 mg/ml.

The lipid may be any lipid which is a functional, biologically activecomponent of naturally occurring HDL or of reconstituted high densitylipoprotein (rHDL). Such lipids include phospholipids, cholesterol,cholesterol-esters, fatty acids and/or triglycerides. Preferably, thelipid is at least one charged or non-charged phospholipid or a mixturethereof.

In a preferred embodiment the rHDL formulation according to the presentinvention comprises a combination of a detergent and a non-chargedphospholipid. In an alternative preferred embodiment the rHDLformulation comprises a charged phospholipid but no detergent at all. Ina further preferred embodiment the rHDL formulation comprises chargedand non-charged lipids as well as a detergent.

As used herein, “non-charged phospholipids”, also called neutralphospholipids, are phospholipids that have a net charge of about zero atphysiological pH. Non-charged phospholipids may be zwitterions, althoughother types of net neutral phospholipids are known and may be used.“Charged phospholipids” are phospholipids that have a net charge atphysiological pH. The charged phospholipid may comprise a single type ofcharged phospholipid, or a mixture of two or more different, typicallylike-charged phospholipids. In some examples, the charged phospholipidsare negatively charged glycophospholipids.

The formulation according to the present invention may also comprise amixture of different lipids, such as a mixture of several non-chargedlipids or of a non-charged lipid and a charged lipid. Examples ofphospholipids include phosphatidylcholine (lecithin), phosphatidic acid,phosphatidylethanolamine (cephalin), phosphatidylglycerol (PG),phosphatidylserine (PS), phosphatidylinositol (PI) and sphinogomyelin(SM) or natural or synthetic derivatives thereof. Natural derivativesinclude egg phosphatidylcholine, egg phosphatidylglycerol, soy beanphosphatidylcholine, hydrogenated soy bean phosphatidylcholine, soy beanphosphatidylglycerol, brain phosphatidylserine, sphingolipids, brainsphingomyelin, egg sphingomyelin, galactocerebroside, gangliosides,cerebrosides, cephalin, cardiolipin and dicetylphospate. Syntheticderivatives include dipalmitoylphosphatidylcholine (DPPC),didecanoyl-phosphatidylcholine (DDPC), dierucoylphosphatidylcholine(DEPC), dimyristoylphosphatidylcholine (DLPC),palmitoyl-oleoylphosphatidylcholine (PMPC),palmitoylstearoyl-phosphatidylcholine (PSPC),dioleoylphosphatidyl-ethanolamine (DOPE), dilauroylphosphatidylglycerol(DLPG), distearoylphosphatidylglycerol (DSPG),dioleoyl-phosphatidylglycerol (DOPG),palmitoyloleoylphosphatidyl-glycerol (POPG), dimyrstolyphosphatidic acid(DMPA), dipalmitoylphosphatidic acid (DPPA), distearoyl-phosphatidicacid (DSPA), dipalmitoylphosphatidylserine (DPPS),distearoylphosphatidylethanolamine (DSPE),di-oleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylserine(DOPS), dipalmitoylsphingomyelin (DPSM) and distearoylsphingomyelin(DSSM). The phospholipid can also be a derivative or analogue of any ofthe above phospholipids. Best results could be obtained withphosphatidylcholine. In another embodiment the lipids in the formulationaccording to the present invention are sphingomyelin and a negativelycharged phospholipid, such as phosphatidylglycerol (e.g. DPPG). Amixture of sphingomyelin and phosphatidylglycerol (particularly DPPG) isspecifically envisaged for use in the invention. In these embodiments,the sphingomyelin and the phosphatidylglycerol may be present in anysuitable ratio, e.g. from 90:10 to 99:1 (w:w), typically 95:5 to 98:2and most typically 97:3.

The formulation according to the present invention typically has alyophilization stabilizer concentration from about 1.0% to about 6.0%e.g. from 1.0, 1.1, 1.2 or 1.3% to 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0%,preferably from about 1.0% to less than 6.0%, e.g. from about 1.0% to5.9% (w/w of rHDL formulation). Preferably from about 3.0% to less than6.0%, e.g. from about 3.0% to 5.9%, preferably from about 4.0 to 5.9%,preferably, from about 4.0% to 5.5%, preferably 4.3 to 5.3%, preferably4.3 to 5.0%, and most preferably from 4.6 to 4.8% (w/w) and in saidformulation the ratio between the apolipoprotein and the lipid ispreferably from about 1:20 to about 1:75, more preferably from about1:45 to about 1:65 (mol:mol). The lyophilization stabilizer ispreferably a sugar (e.g. sucrose), optionally in combination with asugar alcohol such as mannitol or sorbitol, or an amino acid such asproline.

In a preferred embodiment, the rHDL formulation according to the presentinvention has a pH in the range of 6 to 8, preferably within the rangeof 7 to 8. Even more preferably the pH is in the range of 7.3 to 7.7.

In a preferred embodiment of the present invention, the formulation islyophilized. Due to the presence of lyophilization stabilizer,preferably of sucrose, sucrose and mannitol, or sucrose and proline, incombination with the apolipoprotein:lipid ratio, the lyophilisationyields in a stable powder having a long shelf life. This powder may bestored, used directly or after storage as a powder or used afterrehydration to form the reconstituted high density lipoproteinformulation.

The invention may be used for large scale production of reconstitutedhigh density lipoprotein. The lyophilized product may be prepared forbulk preparations, or alternatively, the mixed protein/lipid solutionmay be apportioned in smaller containers (for example, single doseunits) prior to lyophilization, and such smaller units may be used assterile unit dosage forms. The lyophilized formulation can bereconstituted in order to obtain a solution or suspension of theprotein-lipid complex, that is the reconstituted high densitylipoprotein. The lyophilized powder is rehydrated with an aqueoussolution to a suitable volume. Preferred aqueous solutions are water forinjection (WFI), phosphate-buffer saline or a physiological salinesolution. The mixture can be agitated to facilitate rehydration.Preferably, the reconstitution step is conducted at room temperature.

It is well known to the person skilled in the art how to obtain asolution comprising the lipid, and the apolipoprotein, such as describedin WO 2012/000048.

In one preferred embodiment, the invention provides a method ofproducing a rHDL formulation including the step of adding thelyophilization stabilizer to the solution comprising the lipid, and theapolipoprotein until a concentration of from about 1.0% to about 6.0%(w/w of rHDL formulation) is reached, e.g. from 1.0, 1.1, 1.2 or 1.3 to5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In a preferred embodiment, thelyophilization stabilizer is added until a concentration from about 1.0%to less than 6.0% e.g. from about 1.0% to 5.9% is reached. Preferablylyophilization stabilizer is added until a concentration from about 3.0to less than 6.0%, e.g. from about 3.0 to 5.9% is reached. Morepreferably the lyophilization stabilizer is added until a concentrationfrom about 4.0 to 5.5%, particularly 4.3 to 5.3%, more particularly 4.3to 5.0%, and most preferably 4.6 to 4.8% (w/w) is reached. The solutionmay already contain stabilizer.

In preferred embodiments the solution additionally includes a detergentsuch as sodium cholate. In a preferred embodiment the rHDL formulationis manufactured by combining Apo A-I purified from plasma, withphosphatidylcholine (PC) in the presence of sodium cholate and sucroseat a concentration from about 1.0% to about 6.0%, preferably from about1.0% to less than 6.0% w/w to produce disc shaped, non-covalentlyassociated particles (MW approximately 144 kDa).

In particular embodiments the rHDL formulation is comprised of an ApoA-I (recombinant or purified from plasma) and phoshatidylcholinestabilized by cholate and sucrose at a concentration from about 1.0% toabout 6.0% w/w, preferably from about 1.0% to less than 6.0%. Inparticular embodiments the cholate levels are from about 0.5 to about1.5 mg/mL. Preferably the recombinant Apo A-I comprises either a wildtype sequence or the Milano sequence (which when expressed formsdimers).

The lyophilized rHDL formulation of the present invention may be formedusing any method of lyophilization known in the art, including, but notlimited to, freeze drying, i.e. the apolipoprotein/lipid-containingsolution is subjected to freezing followed by reduced pressureevaporation.

The lyophilized rHDL formulations that are provided can retainsubstantially their original stability characteristics for at least 2,4, 6, 8, 10, 12, 18, 24, 36 or more months. For example, lyophilizedrHDL formulations stored at 2-8° C. or 25 ° C. can typically retainsubstantially the same molecular size distribution as measured byHPLC-SEC when stored for 6 months or longer. Particular embodiments ofthe rHDL formulation can be stable and suitable for commercialpharmaceutical use for at least 6 months, 12 months, 18 months, 24months, 36 months or even longer when stored at 2-8° C. and/or roomtemperature.

The rHDL formulation according to the present invention may be used inpreventing or treating a disease, disorder or condition in a human.Suitably, the disease, disorder or condition is responsive toprophylactic or therapeutic administration of the rHDL formulationaccording to the present invention. Examples of such diseases, disordersor conditions include atherosclerosis; cardiovascular disease (e.g.acute coronary syndrome (ACS) such as angina pectoris and myocardialinfarction); or diseases, disorders or conditions such as diabetes thatpredispose to ACS; hypercholesterolaemia (e.g. elevated serumcholesterol or elevated LDL cholesterol) and hypocholesterolaemiaresulting from reduced levels of high-density lipoprotein (HDL), such asbeing symptomatic of Tangier disease.

rHDL formulations according to the present invention may be administeredby any route of administration known in the art. Preferably, rHDLformulations are administered parenterally, such as by intravenous (IV)infusion or injection. In preferred embodiments the rHDL formulationcomprises Apo A-I (recombinant or purified from plasma) which has beenreconstituted to form particles suitable for IV infusion.

The administered dosage of the rHDL formulation may be in the range offrom about 1 to about 120 mg/kg body weight. Preferably, the dosage isin the range of from about 5 to about 80 mg/kg inclusive of 8 mg/kg, 10mg/kg, 12 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, and70 mg/kg dosages. Alternatively delivery can be achieved by fixeddosages of rHDL, that is, in an amount independent of patient bodyweight. Preferred fixed dosages include 0.1-15 g, 0.5-12 g, 1-10 g, 2-9g, 3-8 g, 4-7 g or 5-6 g of apolipoprotein. Particularly preferred fixeddosages include 1-2 g, 3-4 g, 5-6 g or 6-7 g of apolipoprotein.Non-limiting examples of specific fixed dosages include 0.25 g, 0.5 g,1.0 g, 1.7 g, 2.0 g, 3.4 g, 4.0 g, 5.1 g, 6.0 g, 6.8 g and 8.0 g ofapolipoprotein. Accordingly, a vial preferably comprises the lyophilizedrHDL formulation with a protein content of 0.25 g, 0.5 g, 1, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8 or 10 g per vial. More preferably theprotein content is either 0.5, 1, 2, 4, 6, 8, or 10 g per vial.

The invention also provides an apolipoprotein kit comprising one or moreunit doses of the apolipoprotein formulation disclosed herein and one ormore other kit components.

Suitably, the kit is for prophylactically or therapeutically treating adisease, disorder or condition in a human, as hereinbefore described.

Non-limiting examples of one or more other kit components includeinstructions for use; vials, containers or other storage vesselscontaining each of the unit doses; delivery devices such as needles,catheters, syringes, tubing and the like; and/or packaging suitable forsafely and conveniently storing and/or transporting the kit. Preferablythe instructions for use are a label or package insert, wherein thelabel or package insert indicates that the apolipoprotein formulationmay be used to treat a disease or condition such as cardiovasculardisease by administering a fixed dose amount to a human subject in needthereof.

A ‘package insert’ refers to instructions included in commercialpackages of the apolipoprotein formulations, that contains informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such apolipoprotein formations.

For the purposes herein, a ‘vial’ refers to a container which holds anapolipoprotein formulation. The vial may be sealed by a stopperpierceable by a syringe. Generally, the vial is formed from a glassmaterial. The apolipoprotein formulation in the vial can be in variousstates including liquid, lyophilized, frozen etc. The fixed dosageapolipoprotein formulation is preferably stable as turbidity is apreferred measure. A turbidity level of below about 5, 10, 15, 20, or 30NTU can generally be considered a stable dosage apolipoproteinformulation. Turbidity measurements can be taken by incubating theapolipoprotein formulations over time periods such as 0 hr, 2 hr, 4hr, 6hr, 12 hr, 18 hr, 24 hr, 36 hr, 72 hr, 7 days and 14 days at storagetemperatures such as room temperature or 2 to 8° C.

Preferably the apolipoprotein formulation is considered to be stable asa liquid when it is stored for 14 days at room temperature and exhibitsa turbidity of less than about 15 NTU.

The kit may facilitate administration of the apolipoprotein formulationby a health professional or self-administration by a patient orcaregiver.

As used herein, the term “comprising” encompasses “including” as well as“consisting” e.g. a formulation or to a component of a formulation thatis described as “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. The different steps can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

BRIEF DESCRIPTION OF DRAWINGS:

FIG. 1 : Molecular size distribution of formulations containing 5 to 10%w/w sucrose.

FIG. 2A: Direct comparison of molecular size distribution offormulations containing 4 and 7.5% w/w sucrose.

FIG. 2B: Molecular size distribution of formulations containing 1, 2, 3,4 and 7.5% (w/w) sucrose.

FIG. 2C: Molecular size distribution of formulations containing sucroseand proline and 7.5% sucrose.

FIGS. 3A and 3B: LCAT activity for 4 to 10% w/w sucrose formulations.

FIG. 3C: LCAT activity for 1, 2, 3, 4 and 7.5% w/w sucrose formulations.

FIG. 3D: LCAT activity for formulations containing sucrose and proline.

FIG. 4A to 4B: Impact of sucrose concentration on cholesterol efflux.

FIG. 4C: Impact of formulations containing sucrose and proline oncholesterol efflux.

FIGS. 5A to 5H: Turbidity of formulations with different sucroseconcentrations and formulations containing sucrose and proline.

FIG. 6 : Picture of lyo cakes with different sucrose concentration.

FIG. 7 : Picture of lyo cakes with different sucrose concentrations andsucrose and proline.

EXAMPLES Example 1 Preparation of the Samples

To make the samples for the following experiments, sodium cholate (NewZealand Pharmaceuticals) was dissolved in buffer (10 mM NaCl, 1 mM EDTA,10 mM TRIS, pH 8.0) and stirred until clear. Soybean phosphatidylcholine(Phospholipid GmbH) was added to an appropriate volume of the cholateand stirred for 16 h at room temperature. The Apo A-I solution wasdiluted to a protein concentration of 9.0 mg/mL (determined by OD280)with 10 mM NaCl and mixed with an appropriate volume of the lipidsolution to obtain protein to lipid ratio in the range of 1:45 to 1:65.The mixture was stirred at 2-8° C. for 30 min to 16 h. The HDL mimeticswere prepared by cholate dialysis using 1% as a diafiltration buffer.The eluate was concentrated to a protein concentration of 33 to 38 gprotein /L. Sucrose was added to obtain the desired concentration (1%,2%, 3%, 4%, 5%, 6.5%, 7%, 10% w/w). The pH of the solution was adjusted,with 0.2 M NaOH to pH 7.50 ±0.1 after which WFI (water for injection)was added to obtain a protein concentration of 30 mg/mL. The finalformulations were then sterile filtered through a 0.2+0.1 μm filter andfilled into 100 mL glass vials at 1.7 g protein per vial andlyophilized.

In some formulations proline was added to the desired concentration.Proline maintains an isotonic formulation.

Example 2 Molecular Size Distribution

Particle formation was determined using HPLC-SEC and assessed by themolecular size distribution of the various formulations. Size exclusionchromatography (HPLC-SEC) was performed on a Superose 6 HR 10/30 column(GE Healthcare) with 140 mmol/1 NaCl, 10 mmol/1 Na-phosphate, 0.02%NaN3, pH7.4, with a flow rate of 0.5 ml/min. Samples of about 90 _(ug)protein were applied, and elution profiles were recorded at 280 nm.

Little difference was observed for formulations containing 5-10% w/wsucrose in the final formulation (FIG. 1 ), indicating that formulationscontaining % 5% w/w sucrose did not affect particle stability afterreconstitution. FIG. 1 shows a complete chromatogram of (1) internalcontrol, 2: 5% w/w sucrose, 3: 6.5% w/w sucrose, 4: 7.5% w/w sucrose and5: 10% w/w sucrose.

In addition a direct comparison between a 7.5% w/w sucrose formulationand 4% w/w sucrose formulation demonstrated that these formulationsexhibit a similar molecular size distribution (FIG. 2A).

FIGS. 2B and 2C show the results for sucrose concentrations 1, 2, 3, and4% (w/w) and formulations comprising sucrose and proline.

All tested formulations are stable. The sucrose content of 4 to 7.5% w/wwas optimum and did not affect the particle to stability afterreconstitution.

Example 3 LCAT Activation

A measure of the effectiveness of the rHDL particles in variousformulations was determined by measuring the LCAT activity. HDLparticles are capable of sequestering cholesterol from plaques formedalong artery walls or cells by interaction with the ATP-binding cassettetransporter Al (ABCA1). Lecithin-cholesterol acyltransferase (LCAT), aplasma enzyme converts the free cholesterol into cholesteryl ester (amore hydrophobic form of cholesterol), which is then sequestered intothe core of the HDL particle before being transported to the liver to bemetabolized. If the sucrose content in the final formulation affectedthe efficacy of the rHDL particle, LCAT activity would decrease.

The lecithin-cholesterol acyltransferase (LCAT) activity esterificationwas assayed as described by Stokke and Norum (Scand J Clin Lab Invest.1971; 27(1):21-7). 150 μl pooled human plasma (CSL Behring) wasincubated with 10 μl rHDL sample and 150 μl PBS in the presence of 20 μl[4-14C]cholesterol (7.5 μCi/ml) for 1.5 h at 4° C. To initiate theesterification of cholesterol, half of the reaction mixture was placedat 37° C. for 30 min while the other half was further incubated at 4° C.for 30 min (to determine background noise). For both samples thecholesterol and cholesteryl ester is extraction by liquid liquidextraction with n-hexane. The cholesteryl ester was separated fromunesterified cholesterol using a solid phase extraction column (SampliQAmino, Agilent) and measured by scintillation counting. The count rateof the sample stored at 4° C. is subtracted from the count rate of thesample stored at 37° C. The same procedure is also performed with areference sample. The LCAT activity is is expressed as % of theReference sample.

FIGS. 3A and 3B show LCAT activity for 4-10% w/w sucrose formulations.FIG. 3C shows LCAT activity for 1-4% w/w sucrose formulations. Verylittle difference is seen in LCAT activity when the sucrose ranges from5-10% w/w in the final formulation (FIG. 3A), however a slightdecreasing trend is evident when the sucrose is further reduced to 4%w/w (FIG. 3B). FIG. 3D shows LCAT activity for formulations comprisingsucrose and proline. No apparent trend in LCAT activity is observed forformulations containing sucrose and proline. Thus the efficacy of theHDL particle in sucrose/proline formulations is maintained.

Example 4 Cholesterol Efflux

Reverse cholesterol transport (ROT) is a pathway by which accumulatedcholesterol is transported from the vessel wall to the liver forexcretion. Cells efflux free cholesterol to lipid-poor Apo A-I via theABCA1 pathway. The cholesterol efflux assay measures the capacity of HDLto accept cholesterol released from cells. It is anticipated that ifsucrose content affected particle formation and/or integrity,differences would affect cholesterol efflux.

Cholesterol efflux from murine macrophage cell lines J774 and RAW 264.7is highly responsive to cAMP stimulation, which leads to theup-regulation of ABCA1 (Bortnick et. al.,. J Biol Chem. 2000;275(37):28634-40). RAW264.7 cells were obtained from the American TypeCulture Collection (ATCC). Cells were cultured in DMEM (Dulbecco'smodified Eagle's medium, Gibco) supplemented with 10% (v/v) foetal calfserum (FCS, Gibco), 2 mM glutamine, 100 units/mL penicillin and 100μg/mL streptomycin in a humidified CO₂ incubator at 37°. For effluxexperiments, cells were seeded into 24-well plates at a density of0.35×10⁶ cells per well. The following day, cells were labeled with[1,2-³H]cholesterol (1 μCi/mL, GE) in DMEM supplemented with 5% (v/v)FCS. After a labelling period of 36 h, cells were washed with phosphatebuffered saline (PBS) and then incubated in DMEM containing 0.2%fatty-acid-free bovine serum albumin (BSA) in the absence or presence of0.3 mM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt-cAMP(8Br-cAMP) for 16 h to up-regulate ABCA1. Following two washes with PBS,cells were incubated with different cholesterol acceptors in DMEM/ 0.2%fatty-acid-free BSA medium. After 5-6 h of incubation, plates werecentrifuged at 500 g for 10 minutes to remove any floating cells andcellular debris. Radioactivity in cell supernatants was measured byliquid scintillation counting. Total cell-associated [³H]cholesterol wasdetermined after extraction of cells in control wells for at least 30minutes with 0.1 M Triton X-100. Cholesterol efflux was expressed as thepercentage of the radioactivity released from cells into the mediumrelative to the total radioactivity in cells and medium. The differencein efflux between control and 8Br-cAMP-stimulated cells was taken as ameasure of ABCA1-dependent efflux.

FIGS. 4A and 4B show that as sucrose concentration decreases from 7.5%w/w to 4% w/w the cholesterol efflux increased. No apparent differencein cholesterol efflux was observed between the proline containingformulations and the 7.5% sucrose formulation (FIG. 4C).

Example 5 Turbidity

The term turbidity is used to describe the cloudiness or haze in asolution. Strictly, turbidity arises from the multiple scattering eventsof visible light by elements present in the solution. Since turbidityarises from the net scattered light, it depends on the sample pathlength, protein concentration and size of theprotein/aggregates/particles. Given that all reduced sucroseformulations contained the same protein concentration uponreconstitution and were measured with the same path length, differencesin turbidity can be attributed to differences in the size and/or numberof protein/aggregates/particles resulting from the various sucroseformulations.

Turbidity was determined with a LED nephelometer (Hach 2100ANTurbiditimeter, Loveland, Colo.) using formacin as a standard. Resultsare given as relative light scattering (NTU).

Formulations containing 4-10% w/w sucrose produced similar turbidsolutions upon reconstitution (FIGS. 5A & 5B). Sucrose concentrations ofless than 4% showed increased turbidity (FIGS. 5E and 5G). Based onturbidity, sucrose concentrations of 4% (w/w) and above are optimum.

Relative increases in the turbidity of a solution upon is storage, isoften cited as an indication of aggregation in proteinbiopharmaceuticals. FIGS. 5C, 5D, 5F and 5H show that little to noincrease in turbidity are seen upon storage in liquid form, therebyindicating stability of the particles.

Example 6 Lyo Cake Appearance

Sucrose formulations with 4% w/w and 7.5% w/w sucrose produced the moststable lyo cakes (FIG. 6 ).

Sucrose formulations with 1 to 4% w/w, and formulations containingsucrose and proline, also produced stable lyo cakes (FIG. 7 ).

Example 7 Stability of rHDL Formulations

The stability of lyophilized rHDL formulations (prepared as perExample 1) was examined before and after storage (protected from light)at 40 C for 12 weeks. Parameters s tested included pH, turbidity, LCATactivation, HPLC-SEC (aggregate content, % lipoprotein in single peakand its relative retention time) and cholesterol efflux (C-efflux)(Tables 1 & 2). The results indicate that the formulations remain stableover the storage period.

TABLE 1 t = 0 t = 12 weeks 1385.E009.09- 1% 2% 3% 4% 7.5% 1% 2% 3% 4%7.5% 13/40° C. sucrose sucrose sucrose sucrose sucrose sucrose sucrosesucrose sucrose sucrose Turbidity 12.4 10.4 8.63 6.87 6.06 13.7 11.17.24 5.41 5.16 LCAT- 97 102 104 110 111 94 101 100 98 105 activationHPLC - SEC- <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 AggregatesHPLC-SEC - 98.8 99.2 99.5 99.6 99.6 99.3 99.7 99.6 99.6 99.6 Lipoproteinpeak C-efflux (total 110 95 103 119 102 109 85 114 116 84 efflux)

TABLE 2 t = 0 t = 12 weeks 1385.E009.14- 1% sucrose/ 3% sucrose/ 4%sucrose/ 1% sucrose/ 3% sucrose/ 4% sucrose/ 16/40° C. 2.2% proline 1.5%proline 1.2% proline 2.2% proline 1.5% proline 1.2% proline Turbidity8.20 8.48 7.48 8.74 6.14 6.12 LCAT-activation 104 116 109 95 103 99HPLC - SEC <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 Aggregates HPLC-SEC 98.6 99.499.5 99.2 99.5 99.7 Lipoprotein peak C-efflux (total 114 80 97 120 10899 efflux)

1.-53. (canceled)
 54. A reconstituted high density lipoprotein (rHDL)formulation comprising an apolipoprotein, a lipid, and a lyophilizationstabilizer, wherein the ratio between the apolipoprotein and the lipidis from about 1:45 to about 1:65 (mol:mol), and the total concentrationof all lyophilization stabilizers present in the composition is fromabout 1.0% to less than 6.0% (w/w of the rHDL formulation).
 55. The rHDLformulation according to claim 54, wherein the ratio between theapolipoprotein and the lyophilization stabilizer is selected from thegroup consisting of (i) from about 1:1 (w:w) to about 1:7 (w:w); (ii)from about 1:1 (w:w) to about 1:3 (w:w); (iii) from about 1:1 (w:w) toabout 1:2.4 (w:w); and (iv) from about 1:1 (w:w) to less than 1:2 (w:w).56. The rHDL formulation according to claim 54, wherein thelyophilization stabilizer is present in a concentration selected fromthe group consisting of (i) from about 1.0 to 5.9% (w/w); (ii) fromabout 3.0 to 5.9% (w/w); (iii) from about 4.0 to 5.5% (w/w); (iv) from4.3 to 5.3% (w/w); and (v) from 4.6 to 4.8% (w/w).
 57. The rHDLformulation according to claim 54, wherein the lyophilization stabilizeris selected from a sugar, a sugar alcohol, an amino acid, a mixture of asugar and a sugar alcohol, a mixture of a sugar and an amino acid, and amixture of a sugar alcohol and an amino acid.
 58. The rHDL formulationaccording to claim 54, wherein the lyophilization stabilizer comprises asugar selected from the group consisting of a monosaccharide,disaccharide and trisaccharide.
 59. The rHDL formulation according toaccording to claim 54, wherein the lyophilization stabilizer comprises adisaccharide selected from the group consisting of fructose, trehalose,maltose and lactose.
 60. The rHDL formulation according to claim 54,wherein the lyophilization stabilizer comprises a sugar alcohol selectedfrom the group consisting of mannitol, inositol, xylitol, galactitol andsorbitol.
 61. The rHDL formulation according to claim 54, wherein thelyophilization stabilizer comprises an amino acid selected from thegroup consisting of proline, glycine, serine, alanine, lysine,4-hydroxyproline, L-serine, sodium glutamate, lysine hydrochloride,sarcosine, and y-aminobutyric acid.
 62. The rHDL formulation accordingto claim 54, wherein the lyophilization stabilizer comprises a mixtureof a sugar and an amino acid selected from the group consisting of (i)1% sucrose and 2.2% proline (w/w); (ii) 3% sucrose and 1.5% proline(w/w); and (iii) 4% sucrose and 1.2% proline (w/w).
 63. The rHDLformulation according to claim 54, wherein the formulation furthercomprises a detergent.
 64. The rHDL formulation according to claim 63,wherein the detergent comprises sodium cholate.
 65. The rHDL formulationaccording to claim 54, wherein the concentration of the apolipoproteinis from about 5 to about 50 mg/ml.
 66. The rHDL formulation according toclaim 54, wherein the apolipoprotein comprises apolipoprotein A-I (ApoA-I).
 67. The rHDL formulation according to claim 54, wherein theapolipoprotein comprises a fragment of apolipoprotein.
 68. The rHDLformulation according to claim 54, wherein the lipid comprises aphosphatidylcholine.
 69. The rHDL formulation according to claim 54,wherein the apolipoprotein is recombinant Apo A-I, the lipid is amixture of sphingomyelin and phosphatidylglycerol, and thelyophilization stabilizer is a mixture of sucrose and mannitol.
 70. ArHDL formulation according to claim 54, wherein the formulation islyophilized.
 71. A vial comprising the lyophilized rHDL formulationaccording to claim 70, wherein the protein content is 1, 2, 4, 6, 8, or10 g per vial.
 72. A method of producing a rHDL formulation comprisingan apolipoprotein, a lipid, and a lyophilization stabilizer having aratio between the apolipoprotein and the lipid of from about 1:45 toabout 1:65 (mol:mol), comprising adding the lyophilization stabilizer toa solution comprising the lipid and the apolipoprotein to provide atotal concentration of all lyophilization stabilizers present in therHDL formulation of from about 1.0% to less than 6.0% (w/w) is reached.73. A method of preventing or treating a disease, disorder or conditionin a human, comprising administering the rHDL formulation according toclaim 54 to the human to thereby prevent or treat the disease, disorderor condition.